The mechanism of genetic recombination in transformation is the replacement of a single strand sequence in the recipient chromosome by the corresponding sequence of a single strand of donor DNA. The physical association of donor DNA with the recipient genome is paralleled by the appearance of recombinant DNA that carries both donor and recipient genetic markers (Ghei et al., J. Bacteriol, 93, 816-829, 1967). However, entry of transforming DNA into a recipient cell does not ensure transformation; the transforming DNA must be integrated with the host cell's DNA. Early discoveries by Lacks and Hotchkiss (Biochem. Biopys. ACTA, 39, 508-517, 1960) found that cells in different mutant cultures bind to or allow entry of the same amount of wild type DNA, but these cells exhibit different degrees of genetic transformation. Steps subsequent to the binding and entry of transforming DNA, culminating in the integration into the host genome, are important in establishing the degree of transformation; this depends on the individual mutation and marker effects. Physiological conditions, defined as the "competence" of the bacterial cell to undergo transformation, control the cell's ability to bind and allow entry of transforming DNA. The proportion of competent cells can be calculated by genetic analysis--by comparing the frequency observed for double transformation of the unlinked markers (or mutation sites) with the frequency predicted from the product of single transformation frequencies.
In molecular cloning it is desirable to enrich for recombinant plasmids with respect to the regenerated vector in the transformed cell population. For cloning in Escherichia coli the treatment of a linearized vector with alkaline phosphatase is commonly used for such enrichment. Exploration of this approach in S. pneumoniae showed that it was unsuitable for cloning fragments present in heterogeneous mixtures of chromosomal DNA on account of the fate of DNA during entry into cells of transformable Gram-positive species. However, the phosphatase method was applicable for the cloning of homogeneous DNA fragments that are derived, for example, from plasmids or viruses.
A novel method for enrichment of recombinant plasmids containing inserts homologous to the host chromosome is described in the Specific Disclosure. This method is based on the phenomenon of chromosomal facilitation of plasmid establishment (Lopez, et al., J. Bacteriol., 150, 692-701, 1982). Inasmuch as plasmids with homology to the chromosome are transferred at least ten times more frequently than vectors lacking homology, an additional passage of the plasmid population through the host will enrich for recombinants by a similar factor. Although demonstrated here in S. pneumoniae, this method applies to other Gram-positive bacteria, such as Bacillus subtilis, as well.
The study of alkaline phosphatase treatment of the vector is illuminating in several respects. In the process of binding and uptake of transforming DNA in S. pneumoniae and B. subtilis, the donor DNA is converted to single-stranded DNA fragments within the cell. The failure of hemiligated recombinant plasmids--generated by the ligation of an alkaline phosphatase treated vector with heterogeneous DNA fragments--to be established can be attributed to this process. Because of the similarity of their uptake mechanisms to S. pneumoniae, alkaline phosphatase treatment has limited value for cloning in other species of Streptococcus or Bacillus. Construction of recombinant plasmids using a phosphatase treated vector in Streptococcus sanguis has been reported for plasmid fragments, but not for chromosomal DNA (Macrina, et al., J. Bacteriol., 143, 1425-1435, 1980). Inasmuch as alkaline phosphatase treatment of vector is an efficient technique for enrichment of recombinant plasmids in E. coli, it appears that DNA molecules entering the E. coli cell remain intact and double stranded.
The ability of recombinant hemiligated plasmids with heterogeneous inserts to become established in a particular transformable species for which the structure of DNA molecules entering the cell is not known is useful as a diagnostic tool to determine whether the DNA gaining entry has been converted to single-stranded fragments.